Leupeptin

Cells were solubilized with lysis buffer [50 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 0.5% (v/v) NP-40, 50 mM NaF, 1 mM Na₃VO₄, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.25 mM pAPMSF] and centrifuged at 12,000 × g for 20 min. For co-immunoprecipitation assay, cells were sonicated briefly in lysis buffer before centrifugation. The lysates were subjected to immunoprecipitation, SDS-PAGE, and Western blot analyses as described^{2 (link)}.

SaOS2 cells were transfected with the respective siRNAs and then with the respective expression plasmids for VSVG-GFP, VSVG-Myc, VSVG-MT1-MMP, or VSVG-KDELR-Myc with or without expression plasmid for sr-IFT20 or IFT20. To examine the ER-to-cell surface transport, VSVG-GFP-transfected SaOS2 cells were incubated overnight at 40 °C in DMEM containing 1% FBS to accumulate VSVG-GFP at the ER, and then shifted to DMEM containing 1% FBS and 0.1 mg/ml cycloheximide pre-warmed at 32 °C to allow transport through the Golgi. After 30 or 60 min in culture, cell surface proteins were biotinylated with 0.5 mg/ml Sulfo-NHS-ss-biotin (Thermo) in Dulbecco’s PBS (DPBS) for 30 min at 4 °C and quenched with 50 mM NH₄Cl in DPBS for 10 min at 4 °C. After washing with ice-cold DPBS, cells were solubilized with Triton X-100 lysis buffer [25 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.4% (w/v) sodium deoxycholate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.25 mM pAPMSF]. Biotinylated proteins were affinity-purified with streptavidin-Sepharose beads and subjected to SDS-PAGE followed Western blot analyses. ER-to-cis-Golgi and intra-Golgi transport of VSVG and VSVG-MT1-MMP and retrograde transport of VSVG-KDELR have been described previously^{48 (link)}.

Total proteins were extracted from 100 mg of sample using extraction buffer (100 mM Tris-Cl pH8, 150 mM NaCl, 0.6% IGEPAL, 1 mM EDTA, 3 mM DTT with protease inhibitors, PMSF, leupeptin, aprotinin, pepstatin, antipain, chymostatin, Na₂VO₃, NaF, MG132, and MG115. Proteins were separated on a 10% polyacrylamide gel. Immunoblot analysis was carried out using mouse α-GFP (1:2000; Invitrogen) for TuMV GFP and rat α-HA (1:500) antibody for pCas13a. The antigens were detected by chemiluminescence using an ECL-detecting reagent (Thermo Scientific).

Mitophagy was measured in eight-to-ten-week-old male mtKeima mice^{32 (link)} or; C57BL/6J (strain# 000664) injected with Ad-Cox8-EGFP-mCherry (1 × 10⁹ PFU diluted in 200 μl of PBS)^{33 (link)} using intravital microscopy^{52 (link)}. Lysosome inhibitor leupeptin (80 mg/kg), or saline was injected i.p. 16 h prior to the experiment. A second dose of leupeptin (40 mg/kg) or saline was administered 12 h later. Three to five mice were analyzed per experiment. This leupeptin treatment protocol was used prior to isolating and sorting cells into PP and PC populations for immunoblot analysis of mitophagy proteins.

To study endocytic trafficking, HK-2 and NRK-52E cells were, respectively, seeded into tissue culture-treated 35 mm plastic dishes from Ibidi (Glasgow, UK) or uncoated 35 mm glass-bottom dishes (No. 1.5 coverslip, 10 mm glass diameter) from MatTek (Ashland, USA). HK-2 and NRK-52E cells were seeded in 1.5 mL of CM at 60 000 and 75 000 cells per dish, respectively. After 24 h incubation at 37 °C with 5% CO₂, cell culture medium was removed from each dish and replaced with CM containing wheatgerm agglutinin-Alexa Fluor 594 (WGA-AF594; 0.1 or 5 μg mL⁻¹ for HK-2 and NRK-52E cells, respectively), as a physiological marker for endolysosomal content.^{16 (link)} After 4 h, cell culture medium was removed and cells washed with PBS (3 × 1.5 mL). After the final wash, for the short chase experiment, cell culture medium was removed and replaced with freshly prepared filter-sterilised (0.22 μm) CM containing dextrin–OG, colistin–OG or dextrin–colistin-OG (5–10 μg mL⁻¹ OG base) and leupeptin (200 μM). After a further 2 h incubation (pulse), cells were intensively washed with PBS, then, for the short chase experiment, cells were immediately incubated with Hoechst 33342 solution (1 μg mL⁻¹) in CM (without phenol red or leupeptin) for 20 min before imaging. In contrast, for the long chase experiment, after incubation with WGA-AF594 for 4 h, cells were washed and 1.5 mL CM containing leupeptin (200 μM) was added to each well. After 1 h incubation, media was removed and replaced with CM containing filter-sterilised (0.22 μm) dextran-Alexa Fluor 488 (dextran-AF488, as control), dextrin–OG, colistin–OG or dextrin–colistin-OG and leupeptin (200 μM) for 2 h. Subsequently, cells were intensively washed with PBS then CM containing leupeptin (200 μM) was added and the plates incubated at 37 °C with 5% CO₂ overnight. Finally, cells were washed with PBS then incubated with Hoechst 33342 solution (1 μg mL⁻¹) in CM (without phenol red or leupeptin) for 20 min before imaging.
To study accumulation in mitochondria and Golgi in HK-2 cells, the same method was used, except that the WGA-AF594 step was omitted and at the end of the chase period, cells were incubated with Mitotracker™ Red FM (100 nM) or BODIPY™ TR ceramide (5 μM) in CM for 20 min before washing with PBS (3 × 1.5 mL). The protocols used here are summarised in Schemes S1 and 2.†